Fast Track Paper

ASBESTOS AND CIGARETTE SMOKE CAUSE INCREASED DNA STRANDBREAKS AND NECROSIS IN BRONCHIOLAR EPITHELIAL CELLS IN VIVO

MICHAEL JUNG,* WENDELL P. DAVIS,* DOUGLAS J. TAATJES,* A NDREW CHURG,† and BROOKE T. MOSSMAN**Departments of Pathology, University of Vermont College of Medicine, Burlington, VT, USA; and†University of British

Columbia, Vancouver, British Columbia, Canada

(Received10 January2000;Revised24 February2000;Accepted24 February2000)

Abstract—Coexposures to asbestos and cigarette smoke cause increased risks of lung cancer in asbestos workers.Although these carcinogens cause DNA damage to epithelial cells in vitro via generation of reactive oxygen species(ROS), it is unclear whether they cause injury to bronchiolar epithelial cells (i.e., the target cells of lung cancers in vivo).We exposed rats to amosite asbestos, cigarette smoke, and the two agents in combination for 1, 2, and 14 d. Numbersof cells exhibiting DNA strand breaks in comparison to sham rats were then evaluated in lungs using the terminaldeoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end labeling (TUNEL) method and by transmissionelectron microscopy (TEM). Increases in TUNEL-positive, necrotic epithelial cells occurred after exposure to asbestosalone and in an additive fashion after smoke and asbestos in combination. These results indicate that DNA strandbreakage and necrosis are prominent mechanisms of injury by asbestos fibers and cigarette smoke in vivo to epithelialcells of the respiratory tract, thus validating in vitro observations from a number of laboratories. © 2000 ElsevierScience Inc.

INTRODUCTION

Cigarette smoking increases the incidence of asbestos-associated lung cancers in heavily exposed worker pop-ulations (reviewed in [1], but the mechanisms of injuryand cocarcinogenesis by these agents in target cells ofthe lung are unclear. Work by a number of laboratoriesshows that asbestos and cigarette smoke generate ROS aswell as increases in antioxidant enzymes in tracheobron-chial and pulmonary epithelial cells [2–4]. Cigarettesmoke also increases pulmonary retention [5] and uptakeof fibers and particles by tracheal epithelial cells throughoxidant-dependent mechanisms [6], and ROS may beimportant in cell injury and transformation by both as-bestos and cigarette smoke (reviewed in [2,7]). AlthoughDNA damage and deletions by these agents have beendemonstrated biochemically in some experimental mod-els [8,9] and may play a role in alveolar epithelial cellinjury in vitro [10], the phenotypic ramifications of as-bestos and cigarette smoke on bronchial epithelial cells,the progenitor cell types of bronchogenic carcinomas,

have not been elucidated in vivo. The objective of workhere was to determine whether DNA strand breakage isan in vivo endpoint of injury by cigarette smoke andasbestos to epithelial cells of the respiratory tract. More-over, we wanted to determine the relationship betweenpatterns of epithelial cell injury to cell proliferation bythese agents, alone and in combination, as previouslycharacterized in this rodent model [11].

Male Sprague Dawley rats weighing approximately250 g were divided into four treatment groups (n 54/group): (i) clean air (sham); (ii) amosite asbestos alone(International Union Against Cancer reference sampleadministered as a single intratracheal instillation of 2.5mg in 0.5 ml physiologic saline using light halothaneanaesthesia at time 0); (iii) cigarette smoke exposurealone; and (iv) cigarette smoke administered for varioustime periods at 1 h after injection of asbestos (i.e.,combination group). Smoke exposures for 1, 2, or 14 dwere carried out in a nose-only smoking apparatus aspreviously described [11,12]. Each rat was exposed dailyto the whole smoke of seven commercial nonfilter ciga-rettes. All animals were killed 24 h after the last smokeexposure by urethane overdose, and the lungs removedand fixed for 24 h by intratracheal inflation with para-formaldehyde to 10 cm of water pressure. The left lung

Address correspondence to: Dr. B. T. Mossman, University of Ver-mont College of Medicine, Department of Pathology, Medical AlumniBuilding, Burlington, VT 05405, USA; Tel: (802) 656-0382; Fax: (802)656-8892; E-Mail: bmossman@zoo.uvm.edu.

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was dehydrated, embedded in paraffin, and sections cutat 5 mm thickness. Rats exposed to asbestos, cigarettesmoke or the two in combination did not lose weight andappeared to have the same activity patterns as sham rats.

DNA strand breaks were evaluated using the terminaldeoxynucleotidyl transferase (TDT)-mediated dUTP-bi-otin nick end labeling (TUNEL) method, which detects39-OH ends of single-stranded DNA. This techniquedetects both apoptotic and necrotic cells [13]. In studieshere, rat lung sections were deparaffinized in xylene,rehydrated through a series of graded alcohol washes(100, 95, 75, and 50%), and rinsed twice for 3 min inphosphate-buffered saline (PBS). Tissue sections werethen permeabilized with 0.5% Triton-X in PBS for 5 minfollowed by two rinses in PBS. To reduce nonspecificbinding and deproteinize, sections were washed in 0.2 MHCl for 8 min, followed by two rinses in PBS. Toremove endogenous peroxidase activity, sections weretreated with 2% H2O2 in ethanol for 5 min and rinsedtwice in PBS. In the TUNEL reaction mixture, 10ml/section of TUNEL enzyme (terminal deoxynucleotidyltransferase in a reaction buffer of 200 mM cacodylicacid, 200 mM KCl, 1mM ethylenediaminetetraacetate(EDTA), 4 mM m-mercaptoethanol, 50% glycerine, pH6.5) (Boehringer Mannheim, Indianapolis, IN, USA) wasused in combination with 90ml/section of TUNEL label

(Boehringer Mannheim). As a negative control, TUNELlabel (fluorescein-dUTP and unlabeled dUTP in a reac-tion buffer of 200 mM potassium cacodylate, 25 mMTris-HCl, 1 mM CaCl2, 0.25 ng/ml bovine serum albu-min, pH 6.6) was applied in the absence of enzyme. Toprevent evaporation, each section was covered withparafilm and then incubated for 30 min at 37°C, followedby three washes in PBS. Conversion was conducted viaapplication of 75ml antifluorescein antibody (Fab frag-ment from sheep, conjugated with peroxidase; TUNELPOD; Boehringer Mannheim) per section and incubationfor 30 min at 37°C, followed by three washes in PBS.Sections were then stained with diaminobenzidine(DAB)-H2O2 solution for 5 min and rinsed in PBS.Finally, hematoxylin was used as a counterstain, sectionswere dehydrated through a series of graded alcohols (50,75, and 100%), and rinsed in xylene. All rinses andwashes were carried at room temperature unless other-wise specified.

Lung sections were evaluated using an OlympusBX50 upright compound light microscope. Digital im-ages (6403 480 pixels) were captured with a micro-scope-mounted Sony DXC-960/MDLLP modified ccdcamera attached to a frame grabber board in a Sun-SPARC station 5 using the Image Capture module ofIMIX Imagist software (Princeton Gamma Tech, Prince-

Fig. 1. Demonstration of TUNEL-positive nuclei (brown staining indicated by arrows) in epithelial cells in rat lungs: (A) Sham-exposedrat lung at 1 d at 4753 magnification. (B) Asbestos-exposed bronchiolar epithelium at 14 d. (C) Bronchiole exposed to asbestos andcigarette smoke at 1 d. (D) Smoke and asbestos-exposed bronchiole at 14 d. (B–D) are 9503 magnification.

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ton, NJ) on the workstation. Digital image processingand semi-quantitative analysis was accomplished using aFeature Analysis module. Digital images were stored on1 gigabyte optical disks. Hard copy photographs weremade from the digital images with a Mitsubishi CP1000color video dye sublimation printer and publication pho-tographs were made with a Fujix Pictrography 3000video printer. Epithelial cells in distinct membranous(terminal) bronchioles first were evaluated at 4003 mag-nification. For each time point, data were collected foreach animal (n 5 4/group) as follows: the number ofTUNEL-positive cells per unit length from 5 randomlyselected bronchioles on each of two sections/animal wereevaluated and expressed as TUNEL- positive cells perlength of bronchiolar epithelium. Selected tissues werealso examined by TEM to determine if cell death oc-curred by necrosis or apoptosis. Lungs that were alreadyfixed in 4% phosphate-buffered formaldehyde for 24 hwere rinsed 33 (5 mins each) in Millonig’s phosphatebuffer, followed by further fixation in half-strength Kar-novsky’s fixative (2.5% glutaraldehyde, 60% formalde-hyde in Millonig’s buffer) for 2 hrs at 4°C. The tissuewas washed in buffer (33 5 min), followed by postfix-ation in 1% osmium tetroxide in buffer for 30 min at 4°C.Finally, the tissue pieces were dehydrated in a gradedseries of ethanols, and infiltrated and embedded inSpurr’s epoxy resin. Ultrathin sections were cut with adiamond knife, contrasted with uranyl acetate and leadcitrate, and viewed with a JEOL1210 electron micro-scope operated at 60 kV.

In all rat lungs, cells exhibiting DNA strand breakswere predominately observed in the bronchiolar epithe-lium with little to no TUNEL-positive cells in othercompartments of the lung. Sham control animals re-vealed few TUNEL-positive cells in the intact bronchio-lar epithelium (Fig. 1A). However, an occasional super-ficial or sloughing ciliated epithelial cell labeledintensely. In comparison to sham controls, TUNEL-pos-itive epithelial cells, both basal and superficial, werenoted in rat membranous bronchioles exposed to asbes-tos, cigarette smoke or the two in combination (Figs.1B–1D). Frequently, TUNEL-positive sloughing epithe-lial cells were observed in the lumen of the airway. Afterexposure to asbestos and/or cigarette smoke, some alve-olar type II epithelial cells occasionally stained posi-tively by the TUNEL method. These patterns of injuryare consistent with observations in human lungs whereboth cigarette smoke and asbestos exposures have beenshown to produce inflammatory and fibrotic changes inmembranous and respiratory bronchioles.

To quantitate numbers of TUNEL-positive cells in-duced by agents over time, computer-assisted imageanalysis was employed. In comparison to sham controls,numbers of TUNEL-positive epithelial cells in distinct

membranous bronchioles of animals were increased ap-proximately 4-fold in asbestos, smoke and smoke plusasbestos groups at day 1 (Fig. 2). At 14 d, strikingincreases were observed in the group exposed to asbestosalone, and additive effects were seen in smoke andasbestos-exposed rat lungs (p 5 .054). TEM verified thatTUNEL-positive cells reflected primarily necrotic deathof bronchiolar epithelial cells in asbestos and smoke-exposed lungs (Fig. 3). These cells exhibited rupture ofnuclear and organelle membranes and a swollen cyto-plasm with loss of basophilia. Necrotic epithelial cellsoften appeared in the lumen in the presence of neutro-phils or alveolar macrophages containing asbestos fibers(Fig. 4).

Cell death is multifaceted and may occur throughdistinct patterns which are most often classified as ne-crosis or apoptosis [13,14]. Necrosis is defined as deathof cells through external damage which may destroy theintegrity of the plasma membrane. Necrotic debris elicits

Fig. 2. Quantitation of DNA breaks in bronchiolar epithelial cells fromrat lung sections. All membranous bronchioles were evaluated on eachof 2 lung sections from each animal (n 5 4 rats/group/time point).Mean6 SEM of each group. *p 5 .054.

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inflammation and infiltration of phagocytic cells, consis-tent with our observations here and inflammatorychanges in rodent inhalation models of asbestos-inducedlung disease (reviewed in [7]). In contrast, apoptosis orprogrammed cell death is mechanistically distinct, occur-ring with the formation of characteristic apoptotic bodies

in a stereotyped sequence of events. Apoptosis mayoccur spontaneously during normal embryonic develop-ment or in response to chemotherapeutic or other agents[15]. Both H2O2 and cigarette smoke cause apoptosis atlow concentrations and necrosis at high concentrations incells in vitro [16,17]. Moreover, exposure to asbestos invitro yields apoptosis in pleural mesothelial cells whichis ameliorated by antioxidants and inhibitors of mitogen-activated protein kinases [18,19]. However, studies usingtracheal epithelial organ cultures have demonstrated thatnecrosis and exfoliation of superficial cells also occurafter exposure to asbestos [20,21], and recent studiesshow additive or synergistic effects of asbestos andsmoke on necrotic cell death (as assessed by51Cr re-lease) and DNA strand breaks (as measured by alkalineunwinding and ethidium bromide fluorescence) in cul-tured alveolar epithelial cells [10]. Whereas these andother studies provide in vitro evidence that asbestos andcigarette smoke are damaging and genotoxic to cells,data here show that DNA strand breaks occur in lungsafter exposure to these agents in vivo. Moreover, thisdamage culminates in necrotic cell death. Elucidating thetype of cell death by asbestos and cigarette smoke orother types of oxidant injury in lung is important inconsidering that cytoprotective approaches may havedifferential effects on either necrosis or apoptosis. Forexample, overexpression of heat shock proteins (HSP70,HSP27), protects against necrosis, but not apoptosis in-duced by cigarette smoke or H2O2 [16,17].

Necrotic cell injury by asbestos and cigarette smokemay be triggered by DNA damage and can trigger oroccur simultaneously with compensatory hyperplasia[20–22]. Previously, Sekhon et al. [11] documentedchanges in cell proliferation in the lungs of the sameanimals examined here using 59bromodeoxyuridine(BrdU) as an indication of increased DNA synthesis. Ifresults here and the studies by Sekhon and colleagues areexamined comparatively, significant increases in bothBrdU- and TUNEL-positive bronchiolar epithelial cellsare signatures of brief exposures to asbestos and cigarettesmoke. These alterations may reflect initial sites of dep-osition of components of cigarette smoke and asbestosfibers which are translocated peripherally in the lungover time.

CONCLUSION

We show here that DNA strand breaks and necrosisare early molecular and phenotypic changes in bronchio-lar epithelial cells after exposure to asbestos and ciga-rette smoke in vivo. The observation that these endpointsof epithelial injury by asbestos or smoking occurs inlungs of rodents substantiates in vitro experiments by us

Fig. 3. Transmission electron micrograph (TEM) showing necroticvacuolated epithelial cell at 14 days after exposure to asbestos andcigarette smoke. Magnification5 25653.

Fig. 4. Transmission electron micrograph (TEM) of asbestos andsmoke-exposed lung at 14 d indicating sloughed necrotic epithelial cell(arrow) accompanied by infiltration of neutrophils (N) and alveolarmacrophages (M) containing asbestos fibers (arrowhead). Magnifica-tion 5 49403.

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and others suggesting that these lung carcinogens dam-age DNA.

Acknowledgements— We thank Dr. Pamela Vacek, Department ofBiostatistics, for statistical analyses and Laurie Sabens for preparationof the manuscript. Dr. John E. Craighead provided valuable assistancein examining histopathology. Supported by grants #ES06499 and #ES09213 from National Institute of Environmental Health Sciences andgrant #HL39469 from National Heart, Lung, and Blood Institute toB.T.M. and grants from the Medical Research Council of Canada toA.C.

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